Genomic Analysis of Secondary Metabolite Producing Actinomycetes: AcbM is a 2-epi-5-epi-valiolone 7-kinase A dissertation submitted to the Fachbereich Naturwissenschaften II (Chemie/Biologie) Bergische Universität – Gesamthochschule Wuppertal for the degree of Doctor of Natural Sciences (Doctor rerum naturalium) presented by MSc. Chang-Sheng Zhang Wuppertal, May 2002 ACKNOWLEDGEMENTS This work was carried out during the period between September, 1998 to May, 2002, am Lehrstuhl für Chemische Mikrobiologie, Fachbereich Chemie, der Bergischen Universität GH Wuppertal. I am sincerely grateful to: Prof. Dr. W. Piepersberg, for giving me an opportunity to be involved in these two wonderful projects; for his invaluable advice, guidance, support and encouragement throughout these years; for his insightful comments and detailed correctness of this manuscript and for his scientific ideas and vision which would benefit me forever. Prof. Dr. W. Reineke for his acceptance to co-examine this thesis and Prof. Dr. H.-J. Altenbach, Prof. Dr. G. Vogel for their kindness to be co-examiners. Dr. Udo Wehmeier, for his unwavering patience, his everlasting stimulating advice, his fruitful discussions, his enthusiastic encouragement throughout my Ph.D. program and for his critical and insightful comments on this thesis. Dr. A. Stratmann for his useful discussions and guidance in designing and performing experiments on the purification of 2-epi-5-epi-valiolone-7-phosphate. Dr. O. Block for his supply of the cyclitols for enzyme tests. Dr. R. Brückner for making the IC-MS assays. M. Podeschwa for his specialized help in analyzing the NMR data. Dr. J. Altenbuchner for his providing the deletion strains and some plasmids and for his critical reviewing of the manuscript of the gene sponge part. Dr. J.-N. Volff for his expert opinions and comments on the gene sponge part of this thesis. Prof. Dr. Hui-Zhan Zhang, especially for his introducing me into the scientific field of microbiology and molecular biology and for his encouragement during these years. Y. Rockser and E. Sauerbier for their expert technical assistance. All my former and current colleagues for their endless helps and for creating a pleasant and friendly atmosphere in the lab during these years. Dr. Zhong-Yuan Lu and all other friends here in Wuppertal, for their friendship and inspirations, for the happy times spent together. My parent, for all. CONTENTS i CONTENTS Contents i Abbreviations vii Summary ix Zusammenfassung x 1. Introduction I – Gene Sponge 1 1.1: Taxonomy, ecology and genetic features of the genus Streptomyces 1 1.2: Assessing the microbial and genetic diversity in soil 3 1.3: Natural gene transfer in soil 5 1.4: Approaches to make use of the genetic diversity in soil 8 1.4.1: The cloning approach 8 1.4.2: The in vivo "gene sponge" approach 10 1. Introduction II – Acarbose Biosynthesis 1 1.5: The genus Actinoplanes 12 1.6: Structure and pharmaceutical applications of acarbose 13 1.7: The acb-gene cluster for acarbose production in Actinoplanes sp. SE50/110 15 1.8: The acarbose metabolism in Actinoplanes sp. SE50/110 19 1.9: Aim of this work 23 2: Material and Methods 24 2.1: Chemicals and enzymes 24 2.2: Various materials 25 2.3: Bacterial strains, vectors and recombinant plasmids 26 2.3.1: Bacterial strains 26 2.3.2: Vectors 28 2.3.3: Recombinant plasmids 28 2.3.4: Recombinant plasmids constructed in this study 29 2.4: Oligonucleotides 32 Dissertation C.-S. Zhang, BUGH Wuppertal CONTENTS ii 2.5: Media and buffers 33 2.5.1: Media for culturing E. coli 33 2.5.2: Media for culturing bacteriophage 33 2.5.3: Media for culturing actinomycetes 33 2.5.4: Buffers 36 2.6: Antibiotics 41 2.7: Growth and maintenance of bacterial strains 41 2.7.1: Growth and maintenance of E. coli 41 2.7.2: Growth and maintenance of Streptomyces cultures 42 2.7.3: Growth of Actinoplanes sp. cultures 42 2.7.4: Crosses (mating) between Streptomyces species 42 2.8: Environmental applications 42 2.8.1: Preparation of soil for use in microcosms 42 2.8.2: Inoculation of soil microcosms 43 2.8.3: Enumeration of viable propagules from soil 43 2.9: In vitro manipulation of DNA 44 2.9.1: Preparation of plasmid DNA from E. coli 44 2.9.2: Preparation of plasmid DNA from streptomycetes 44 2.9.3: Extraction of genomic DNA from actinomycetes 45 2.9.4: Preparation of bacteriophage DNA 45 2.9.5: Restriction endonuclease digestion of DNA 45 2.9.6: Recovery of DNA fragments from agarose gels 45 2.9.7: Removal of phosphate groups from DNA 46 2.9.8: Generation of blunt ends using the Klenow fragment of DNA polymerase I 46 2.9.9: Ligation of DNA fragments 46 2.9.10: Quantitation of DNA 46 2.9.11: DNA sequencing 47 2.9.12: Southern capillary blotting 47 2.9.13: Labeling of DNA fragments with [32P] 47 2.9.14: DNA-DNA hybridization 47 2.9.15: Autoradiography 48 Dissertation C.-S. Zhang, BUGH Wuppertal CONTENTS iii 2.10: Preparation of competent E. coli cells 48 2.11: Transformation of E. coli strains 48 2.12: Preparation of protoplasts from streptomycetes 49 2.13: Transformation of streptomycete protoplasts with plasmid DNA 49 2.14: Gel electrophoresis of DNA 49 2.14.1: Agarose gel electrophoresis 49 2.14.2: Pulsed field gel electrophoresis (PFGE) 50 2.15: Polymerase chain reactions (PCR) 50 2.15.1: General PCR conditions 50 2.15.2: PCR amplification of specific gene fragments 52 2.15.3: Random amplified polymorphic DNA (RAPD) PCR amplification 52 of specific gene fragments 2.16: SDS polyacrylamide gel electrophoresis (SDS-PAGE) 52 2.17: Heterologous gene expression in E. coli 53 2.17.1: Heterologous gene expression under the control of the T7 promoter 53 2.17.2: Heterologous gene expression under the control of the rha promoter 53 2.18: Heterologous gene expression in S. lividans 66 53 2.18.1: Heterologous gene expression under the control of the ermE-up promoter 53 2.18.2: Heterologous gene expression under the control of the tipA promoter 54 2.19: Cell-free extracts of E. coli and Streptomyces 54 2.20: Cell-free extracts of Actinoplanes sp. SE50 54 2.21: Determination of protein concentration 54 2.22: Western blotting and immuno-detection of proteins 55 2.23: Thin-layer chromatography (TLC) 55 2.24: Purification of His-tag AcbM proteins 55 2.25: Determination of the activity of AcbK 55 2.26: Determination of a 1-epi-valienol kinase activity 56 2.27: Enzymatical synthesis of 2-epi-5-epi-valiolone 56 2.28: Determination of the activity of AcbM 57 2.29: Determination of the activity of AcbO 57 2.30: Purification of 2-epi-5-epi-valiolone 57 2.31: Purification of 2-epi-5-epi-valiolone-7-phosphate 58 Dissertation C.-S. Zhang, BUGH Wuppertal CONTENTS iv 2.32: Ion chromatography and mass spectrometry (IC-MS) 58 2.33: Determination of optical rotations of 2-epi-5-epi-valiolone-7-phosphate 60 2.34: NMR spectrometry 60 2.35: Computer programs 61 3: Result I - Gene Sponge 62 3.1: An new approach for exploring of natural products (bioactive 62 secondary compounds) from soil – use of in vivo "genetic sponges" for uptake of expressible genetic materials 3.2: Characterization of four deletion strains 62 3.2.1: Description of four S. lividans 66 strains with large deletions 62 at the chromosomal ends 3.2.2: Phenotypic behavior of the 4 selected recipient strains in plate cultures 63 3.2.3: Regeneration and transformation of protoplasts of the selected recipient strains 64 3.2.4: Antibiotic resistance pattern of the recipient strains 64 3.3: Survival of S. lividans 66 strains in sterile soil 65 3.4: Transfer of a non-conjugative plasmid pIJ702 in sterile soil 66 3.4.1: Detection of plamid transfer in sterile amended soil 66 3.4.2: Characterization of transconjugants from the amended sterile soil 67 3.5: Possibility of transfer of chromosomal markers in sterile soil 69 3.6: Possibility of gene transfer in nonsterile soil 71 3.6.1: Survival of S. lividans Jni13C1 in nonsterile soil 71 3.6.2: Characterization of some Streptomyces species isolated from nonsterile soil 72 3.6.2.1: Strain characterization by PCR 73 3.6.2.2: Random amplified polymorphic DNA (RAPD) analysis 74 3.6.2.3: Identification of 16S rRNA operons and comparison of the restriction 75 fragment length polymorphisms (RFLPs) 3.6.2.4: Genomic analysis by pulsed field gel electrophoresis (PFGE) 76 3.7: Mobilization of chromosomal markers by the conjugative plasmid pIJ903 77 3.7.1: The conjugative plasmid pIJ903 77 3.7.2: Mating experiments with S. lividans 66 strains 78 3.7.3: Characterization of some SmRCmlR strains from matings 79 between S. lividans 66 strains Dissertation C.-S. Zhang, BUGH Wuppertal CONTENTS v 3.7.4: Chromosome organization of the recombinant strains 85 4: RESULTS II – Acarbose Biosynthesis 90 4.1: Analysis of the genome of Actinoplanes sp. SE50/110 by PFGE 90 4.2: Heterologous expression of the AcbK, M, L, N, O and AcbU proteins 94 4.2.1: The AcbK, AcbM, AcbL, AcbN, AcbO and AcbU proteins 94 4.2.2: The general strategy for the expression of the selected Acb-proteins 96 4.2.3: Heterologous expression of AcbK, AcbM, AcbN, AcbO and AcbU 96 in E. coli K12 and in S. lividans 66 4.3: Characterization of the AcbK protein and the applications thereof 100 4.3.1: Phosphorylation of acarbose by heterologously expressed AcbK 100 4.3.2: Phophorylation assays using AcbK and cyclitols 101 4.3.3: Heterologous production of acarbose in S.